1. Field of the Invention
The invention generally relates to the field of Raman spectroscopy and, more specifically, to porous substrates suitable for use in Raman spectroscopy apparatus.
2. Brief Description of Related Technology
Spectroscopy is an analytical technique useful to measure the radiant energy absorbed or emitted by a substance in response to excitation by an external energy source, and to translate that measurement into meaningful spectra. Interpretation of the spectra is useful to determine fundamental information about the substance, such as, for example, its composition, atomic and molecular energy levels, molecular structures and their geometries, chemical bonding, and interactions between molecules. This interpretation generally is carried out by comparing the spectra obtained from an unknown substance to the spectrum of a known substance. Such comparisons provide a basis from which a skilled artisan can determine the chemical composition and chemical structure of the unknown substance.
The types of absorption and emission spectroscopy are usually identified by reference to the associated wavelength, such as, for example, gamma-ray, infrared (IR), microwave, radiofrequency, ultraviolet (UV), visible, and x-ray. Highly-specialized techniques have been developed since the inception of spectroscopic analysis in the 19th Century, including, for example, dynamic reflectance spectroscopy, electron paramagnetic resonance, gamma-ray spectroscopy, IR spectroscopy, laser, microwave, nuclear magnetic resonance, nuclear quadrupole resonance, and Raman spectroscopy. See generally, Hawley's Condensed Chemical Dictionary, 12th ed., Van Nostrand Reinhold, N.Y., p. 1039 (1997).
One particular spectroscopic technique, known as Raman spectroscopy, is based on the detection of optical energy (e.g., light) that has been scattered by a substance (e.g., a molecule) when excited by an external energy source (e.g., a laser). This scattering is commonly known as the “Raman effect.” When exciting optical energy of a single wavelength (e.g. monochromatic light) interacts with a molecule, for example, the optical energy scattered by the molecule contains small amounts of optical energy having wavelengths different from that of the incident, exciting optical energy. The wavelengths of the scattered optical energy are characteristic of the molecule's structure, and the intensity of the scattered optical energy is related to the concentration of the molecule. The wavelengths are separated by a spectrometer and detected by a detector to provide a Raman spectrum. The output of the detector can be interpreted with the aid of a data processor (e.g., a computer). Each different molecule has its own unique Raman spectrum, which can be used for both qualitative (e.g., identification) and quantification (e.g., determination of concentration) purposes.
Historically, Raman spectroscopy has been a useful spectrochemical technique for chemists in characterizing the chemical make-up of various substances and in identifying unknown molecules. Raman spectroscopy is a technique that is complementary to IR spectroscopy. Due to differences in the spectroscopic selection rules, each is sensitive to different components of a given sample. For example, IR spectroscopy generally is more sensitive to polar bonds (e.g., oxygen-hydrogen bonds), while Raman spectroscopy is more sensitive to vibrations of carbon backbone structures and symmetrical bonds (e.g., C=C groups). Using both spectroscopic techniques to characterize a particular sample may provide information on the sample's chemical composition that might not be obtainable using either of the techniques alone.
More recently, Raman spectroscopy has been used in the biological and other life-sciences areas including, but not limited to, analyses of the stratum corneum in human skin in relation to the administration of therapeutic agents, cancer diagnosis, corneal dehydration in relation to impaired visual acuity, characterization of gallstones and kidney stones, diagnosis of Alzheimer's disease, diagnosis of metabolic disorders by taking Raman spectra of hair and nails, hard tissue implant biocompatibility and in vivo recovery characteristics, imaging of cells (e.g., carotenoids in lymphocytes), and quantitative histochemical analysis of human arteries. Raman spectroscopy also has been implicated as a useful technique in DNA sequencing and in deciphering the human genome. The need for powerful and costly laser sources for excitation and other prohibitively costly equipment in these and other biological and chemical analyses limit the practicality of conventional Raman spectroscopy apparatus.
Conventional Raman spectroscopy of an unpurified sample may detect that the sample gives a broad optical emission signal, much of which is attributable to undesired background fluorescence. Background fluorescence also can be attributed to known fluorophores, trace amounts of adventitious fluorescent impurities in the sample, and from the substrate on which the sample is analyzed. In Raman spectroscopy, such background fluorescence is undesired because it drowns out the relatively weak Raman signal(s) attributable to the target molecule.
One approach to dealing generally with the undesired background fluorescence is to perform background subtractions to discount this fluorescence from the obtained spectrum. However, because Raman signals oftentimes are so weak relative to the background fluorescence, it is difficult to make meaningful and accurate determinations even when discounting the background fluorescence. This difficulty is only exacerbated where the target molecule is present in very low concentrations. Another approach is to avoid the fluorescence by utilizing an excitation energy source in the near-infrared (NIR) region in the absence of electronic absorption and emission transitions. This approach, however, does not permit the resonance effect of Raman spectroscopy to be utilized for many compounds and also suffers from low sensitivity owing to the inverse fourth-power law dependence of non-resonant Raman scattering. Yet another approach is to utilize an excitation energy source well below the fluorescence emission in the UV region. Though this approach desirably permits much higher cross-sections for Raman scattering than in the NIR region, it often leads to resonance enhancement of several constituents of the substance simultaneously, which is undesirable when trying to detect and/or characterize molecules present at low concentration or where molecular selectivity is desired. Other approaches include utilizing surface-enhanced Raman spectroscopy (SERS), shifted excitation Raman difference spectroscopy (SERDS), polarization modulation, shifted spectra, Fourier transform filtering, and temporal gating. See generally, Matousek el al. (2002) J. Raman Spectrosc. 33: 238-242. High expense, high complexity, and/or low reproducibility, however, are undesirable characteristics of each of these approaches.
An approach to combating undesired background fluorescence attributable to the substrate is to utilize a substrate that does not generate interfering signals and/or a substrate having a microcrystalline surface. Such substrates, however, must be of high purity and, thus, are more expensive. Even where one is able to afford substrates having the desired microcrystalline surface, such substrates can only be manufactured to a certain size and, therefore, Raman spectroscopy applications are limited by such size limitations. To diminish the effects of background fluorescence caused by the substrate, the target molecule(s) may be floated in air or water to spatially separate the target molecule(s) from the substrate material —this permits a spatial separation of the Raman signal(s) of the molecule(s) from the interfering signal(s) generated from the substrate. This approach, however, undesirably includes the additional steps of utilizing a liquid or gas to spatially separate the target molecule(s) from the substrate and accompanying associated equipment.